Dataset Information

Mechanically unfolding protein L using a laser-feedback-controlled cantilever.

ABSTRACT: Force spectroscopy using the atomic force microscope (AFM) can yield important information on the strength and lifetimes of the folded states of single proteins and their complexes when they are loaded with force. For example, by mechanically unfolding concatenated proteins at different velocities, a dynamic force spectrum can be built up that allows reconstruction of the energy landscape that the protein traverses during unfolding. To characterize fully the unfolding landscape, however, it is necessary both to explore the entire force spectrum and to characterize each species populated during unfolding. In the conventional AFM apparatus, force is applied to the protein construct through a compliant cantilever. This limits the dynamic range of the force spectrum that can be probed, and the cantilever recoil after unfolding may mask the presence of metastable intermediates. Here, we describe to our knowledge a new technique-constant-deflection AFM-in which the compliance of the AFM cantilever is removed. Using this technique, we show that protein L exhibits a more complex unfolding energy landscape than previously detected using the conventional technique. This technique is also able to detect the presence of a refolding intermediate whose formation is otherwise prevented by cantilever recoil.

SUBMITTER: Crampton N

PROVIDER: S-EPMC3072654 | biostudies-other | 2011 Apr

REPOSITORIES: biostudies-other

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Mechanically unfolding protein L using a laser-feedback-controlled cantilever.

Crampton Neal N Alzahrani Khalid K Beddard Godfrey S GS Connell Simon D SD Brockwell David J DJ

Biophysical journal 20110401 7

Force spectroscopy using the atomic force microscope (AFM) can yield important information on the strength and lifetimes of the folded states of single proteins and their complexes when they are loaded with force. For example, by mechanically unfolding concatenated proteins at different velocities, a dynamic force spectrum can be built up that allows reconstruction of the energy landscape that the protein traverses during unfolding. To characterize fully the unfolding landscape, however, it is n ...[more]

PMID: 21463594

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Project description:Precise quantification of the energetics and interactions that stabilize membrane proteins in a lipid bilayer is a long-sought goal. Toward this end, atomic force microscopy has been used to unfold individual membrane proteins embedded in their native lipid bilayer, typically by retracting the cantilever at a constant velocity. Recently, unfolding intermediates separated by as few as two amino acids were detected using focused-ion-beam-modified ultrashort cantilevers. However, unambiguously discriminating between such closely spaced states remains challenging, in part because any individual unfolding trajectory only occupies a subset of the total number of intermediates. Moreover, structural assignment of these intermediates via worm-like-chain analysis is hindered by brief dwell times compounded with thermal and instrumental noise. To overcome these issues, we moved the cantilever in a sawtooth pattern of 6-12 nm, offset by 0.25-1 nm per cycle, generating a "zigzag" force ramp of alternating positive and negative loading rates. We applied this protocol to the model membrane protein bacteriorhodopsin (bR). In contrast to conventional studies that extract bR's photoactive retinal along with the first transmembrane helix, we unfolded bR in the presence of its retinal. To do so, we introduced a previously developed enzymatic-cleavage site between helices E and F and pulled from the top of the E helix using a site-specific, covalent attachment. The resulting zigzag unfolding trajectories occupied 40% more states per trajectory and occupied those states for longer times than traditional constant-velocity records. In total, we identified 31 intermediates during the unfolding of five helices of EF-cleaved bR. These included a previously reported, mechanically robust intermediate located between helices C and B that, with our enhanced resolution, is now shown to be two distinct states separated by three amino acids. Interestingly, another intermediate directly interacted with the retinal, an interaction confirmed by removing the retinal.

Dataset Information

Mechanically unfolding protein L using a laser-feedback-controlled cantilever.

Publications

Mechanically unfolding protein L using a laser-feedback-controlled cantilever.

Similar Datasets

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